Method of modifying the release profile of sustained release compositions

ABSTRACT

The present invention relates to a method for the sustained release in vivo of a biologically active agent comprising administering to a subject in need of treatment an effective amount of a sustained release composition comprising a biocompatible polymer having the biologically active agent incorporated therein, and a bisphosphonate wherein the bisphosphonate compound is present in an amount sufficient to modify the release profile of the biologically active agent from the sustained release composition. Pharmaceutical compositions suitable for use in the method of the invention are also disclosed.

BACKGROUND OF THE INVENTION

[0001] Many illnesses or conditions require administration of a constantor sustained level of a medicament or biologically active agent toprovide the most effective prophylactic or therapeutic. This may beaccomplished through a multiple dosing regimen or by employing a systemthat releases the medicament in a sustained fashion.

[0002] Attempts to sustain medication levels include the use ofbiodegradable materials, such as polymeric matrices, containing themedicament. The use of these matrices, for example, in the form ofmicroparticles or microcarriers, provides sustained release ofmedicaments by utilizing the inherent biodegradability of the polymer.The ability to provide a sustained level of medicament can result inimproved patient compliance.

[0003] However, these sustained release devices can exhibit high releaseof active agent over the first twenty-four hours, often referred to as aburst. In some instances this burst can result in an undesirableincrease in the levels of biologically active agent and minimal releaseof agent thereafter. In addition, due to the high solution concentrationof medicament within and localized around these sustained releasedevices, the medicament can aggregate thereby increasing immunogenicityin vivo and interfering with the desired release profile for themedicament.

[0004] Therefore, a need exists to exert additional control over therelease profile of sustained release compositions by, for example,reducing the burst of agent and/or providing an improved release such asa longer period of release.

SUMMARY OF THE INVENTION

[0005] The present invention is based upon the unexpected discovery thatthe release profile of a biologically active agent from a sustainedrelease composition comprising a biocompatible polymer and thebiologically active agent incorporated therein can be modified suc as byprolonging the period of release of agent when a bisphosphonate compoundis co-administered.

[0006] Accordingly, the present invention relates to a method for thesustained release in vivo of a biologically active agent comprisingadministering to a subject in need of treatment an effective amount of asustained release composition comprising a biocompatible polymer havingthe biologically active agent incorporated therein, and a bisphosphonatewherein the bisphosphonate is present in an amount sufficient to modifythe release profile of the biologically active agent from the sustainedrelease composition.

[0007] In one embodiment, the bisphosphonate compound can beco-incorporated into the sustained release composition comprising thebiocompatible polymer and the biologically active agent incorporatedtherein.

[0008] In another embodiment, the bisphosphonate compound can beseparately incorporated into a second biocompatible polymer. Thebiocompatible polymer can be the same or different from the firstbiocompatible polymer which has the biologically active agentincorporated therein.

[0009] In yet another embodiment, the bisphosphonate compound can bepresent in an unencapsulated state but comingled with the sustainedrelease composition. For example, the bisphosphonate can be solubilizedin the vehicle used to deliver the sustained release composition.Alternatively, the bisphosphonate compound can be present as a solidsuspended in an appropriate vehicle. Further, the bisphosphonate can bepresent as a powder which is comingled with the sustained releasecomposition.

[0010] The invention described herein also relates to pharmaceuticalcompositions suitable for use in the invention. In one embodiment, thepharmaceutical composition comprises a sustained release compositioncomprising a biocompatible polymer having an effective amount of abiologically active agent incorporated therein, and an amount ofbisphosphonate compound sufficient to modify the release profile of thebiologically active agent from the sustained release composition.

[0011] In one embodiment, the bisphosphonate compound can beco-incorporated into the sustained release composition comprising thebiocompatible polymer and the biologically active agent incorporatedtherein.

[0012] In another embodiment, the pharmaceutical composition comprisesthe sustained release composition comprising a first biocompatiblepolymer having incorporated therein an effective amount of abiologically active agent and a second biocompatible polymer havingincorporated therein an amount of bisphosphonate which modifies therelease profile of the biologically active agent from the first polymer.In a particular embodiment, the first and second polymers are the sametype of polymer. In another embodiment, the first and second polymersare different.

[0013] In yet another embodiment, the bisphosphonate compound can bepresent in the pharmaceutical composition in an unencapsulated state.For example, the bisphosphonate compound can be comingled with thesustained release composition. In one embodiment, the bisphosphonate canbe solubilized in the vehicle used to deliver the pharmaceuticalcomposition. Alternatively, the bisphosphonate compound can be presentas a solid suspended in an appropriate vehicle useful for delivering thepharmaceutical composition. Further, the bisphosphonate can be presentas a powder which is comingled with the sustained release composition.

[0014] Without being bound by a particular theory, it is believed thatat least in part the effects of the bisphosphonates can be related to areduction in the amount of inflammatory cellular reaction which canoccur in the area of administration of the sustained releasecomposition. This reaction, although clinically insignificant, is wellcharacterized as a foreign body response, and can be realized with mostforeign materials.

[0015] The present invention also relates to a composition for thesustained release of bisphosphonates. The sustained release compositioncomprises a biocompatible polymer matrix having a therapeuticallyeffective amount of bisphosphonate incorporated therein. Further, theinvention relates to a method for the sustained release in vivo of abisphosphonate compound comprising administering to a subject in need oftreatment a therapeutically effective amount of a sustained releasecomposition comprising a biocompatible polymer and a bisphosphonatecompound.

[0016] In a particular embodiment, administration of the sustainedrelease composition comprising a biocompatible polymer and abisphosphonate can be to a joint, for example, the articular space of ajoint. For example, the sustained release composition can beadministered to the articular space of the knee, shoulder, ankle, hipetc . . .

[0017] The sustained release composition of the invention comprising abiocompatible polymer and a bisphosphonate compound can be used for thetreatment of diseases associated with bone resorption or jointinflammation. For example, the sustained release composition having abiocompatible polymer and a bisphosphonate compound incorporated thereincan be suitable for use as a treatment for rheumatoid arthritis,osteoporosis or Paget's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings.

[0019]FIG. 1 is a plot of the serum EPO levels (mU/mL) versus time indays post administration of the EPO-containing andbisphosphonate-containing microparticles of the invention.

[0020]FIG. 2 is a plot of % hematocrit versus time in days postadministration of the EPO-containing and bisphosphonate-containingmicroparticles of the invention.

[0021]FIG. 3 is a plot of the serum EPO levels (mU/mL) versus time indays post administration of the EPO-containing andalendronate-containing microparticles of the invention.

[0022] FIGS. 4A-4C are plots of % hematocrit versus time in days postadministration of the EPO-containing and alendronate-containingmicroparticles of the invention.

[0023]FIG. 5 is a plot of the serum EPO levels (mU/mL) versus time indays post administration of microparticles containing EPO andpamidronate co-encapsulated (1% and 10% load).

[0024]FIG. 6 is a plot of % hematocrit versus time in days postadministration of microparticles containing EPO and pamidronateco-encapsulated (1% and 10% load).

DETAILED DESCRIPTION OF THE INVENTION

[0025] A description of preferred embodiments of the invention follows.

[0026] The present invention relates to a method for the sustainedrelease in vivo of a biologically active agent comprising administeringto a subject in need of treatment an effective amount of a sustainedrelease composition comprising a biocompatible polymer having thebiologically active agent incorporated therein, and a bisphosphonatewherein the bisphosphonate is present in an amount sufficient to modifythe release profile of the biologically active agent from the sustainedrelease composition.

[0027] In one embodiment, the bisphosphonate compound can beco-incorporated into the sustained release composition comprising thebiocompatible polymer and the biologically active agent incorporatedtherein.

[0028] In another embodiment, the bisphosphonate compound can beseparately incorporated into a second biocompatible polymer. Thebiocompatible polymer can be the same or different from the firstbiocompatible polymer which has the biologically active agentincorporated therein.

[0029] In yet another embodiment, the bisphosphonate compound can bepresent in an unencapsulated state but comingled with the sustainedrelease composition. For example, the bisphosphonate can be solubilizedin the vehicle used to deliver the sustained release composition.Alternatively, the bisphosphonate compound can be present as a solidsuspended in an appropriate vehicle. Further, the bisphosphonate can bepresent as a powder which is comingled with the sustained releasecomposition.

[0030] Bisphosphonates are a group of synthetic pyrophosphatescharacterized by a P-C-P type backbone. The bisphosphonates are potentinhibitors of bone resorption and ectopic calcification. In general thebisphosphonates can be represented by Formula I:

[0031] wherein,

[0032] R₁ is independently, H, alkyl, aryl or heteroaryl;

[0033] X is H, —OR₁ or halogen;

[0034] R₂ is H, O, S, N, (CH₂)_(n), branched alkylene, branched orstraight alkenylene or alkynylene;

[0035] n is an integer from about 0 to about 18;

[0036] Y is H, R₁, halogen, amino, cyano or amido group.

[0037] As used herein, “alkyl” refers to a straight chain or branched,substituted or unsubstituted C₁-C₁₈ hydrocarbon group. Examples ofsuitable alkyl groups include, but are not limited to, methyl, ethyl,propyl, butyl, pentyl, isopropyl, isobutyl, and tert-butyl. As usedherein, “halogen” refers to chlorine, bromine, iodine and fluorine. Theterm “aryl” as used herein refers to unsubstituted and substitutedaromatic hydrocarbons. The term “heteroaryl” as used herein refers tounsubstituted or substituted aryl groups wherein at least one carbon ofthe aryl group is replaced with a heteroatom (e.g., N, O or S). Suitablesubstituents, include, for example, but are not limited to, halogen,—OH, alkoxy, amino, amido, —SH, cyano, —NO₂, —COOH, —COH, —COOR₁.

[0038] A number of geminal bisphosphonates such as those shown below arecurrently used for the treatment of moderate to severe Paget's diseaseand hypercalcemia associated with malignant neoplasms, treatment ofosteolytic bone lesions associated with multiple myeloma and treatmentof osteoporosis.

[0039] Bisphosphonates suitable for use in the invention include thosedescribed in U.S. Pat. No. 4,705,651, U.S. Pat. No. 4,327,039, US.Patent No. 5,312,954 and U.S. Pat. No. 5,196,409 to Breuer et al., U.S.Pat. No. 5,412,141 to Nugent, U.S. Pat. Nos. 4,922,007 and 5,019,651 toKieczykowski et al., U.S. Pat. No. 5,583,122 to Benedict et al., U.S.Pat. No. 6,080,779 to Gasper et al., U.S. Pat. No. 6,117,856 toBenderman et al., U.S. Pat. No. 6,162,929 to Foricher et al. and U.S.Pat. No. 5,885,473 to Papapoulos et al. the entire content of all ofwhich are hereby incorporated by reference.

[0040] “Patient” as that term is used herein refers to the recipient ofthe treatment. Mammalian and non-mammalian patients are included. In aspecific embodiment, the patient is a mammal, such as a human, canine,murine, feline, bovine, ovine, swine or caprine. In a preferredembodiment, the patient is a human.

[0041] The term “sustained release composition” as defined herein,comprises a biocompatible polymer having incorporated therein at leastone biologically active agent. Suitable biocompatible polymers, can beeither biodegradable or non-biodegradable polymers or blends orcopolymers thereof, as described herein.

[0042] Typically, the sustained release composition can contain fromabout 0.01% (w/w) to about 50% (w/w) of the biologically active agent(dry weight of composition). The amount of agent used will varydepending upon the desired effect of the agent, the planned releaselevels, and the time span over which the agent will be released. Apreferred range of agent loading is between about 0.1% (w/w) to about30% (w/w) agent. A more preferred range of agent loading is betweenabout 0.5% (w/w) to about 20% (w/w) agent.

[0043] The sustained release compositions of this invention can beformed into many shapes such as a film, a pellet, a rod, a filament, acylinder, a disc, a wafer or a microparticle. A microparticle ispreferred. A “microparticle” as defined herein, comprises a polymercomponent having a diameter of less than about one millimeter and havinga biologically active agent dispersed therein. A microparticle can havea spherical, non-spherical or irregular shape. Typically, themicroparticle will be of a size suitable for injection. A preferred sizerange for microparticles is from about one to about 180 microns indiameter.

[0044] As defined herein, a sustained release of biologically activeagent is a release of the agent from a sustained release composition.The release occurs over a period which is longer than that period duringwhich a therapeutically significant amount of the biologically activeagent, would be available following direct administration of a solutionof the biologically active agent. It is preferred that a sustainedrelease be a release of biologically active agent which occurs over aperiod of greater than two days. A sustained release of biologicallyactive agent, from a sustained release composition can be a continuousor a discontinuous release, with relatively constant or varying rates ofrelease. The continuity of release and level of release can be affectedby the type of polymer composition used (e.g., monomer ratios, molecularweight, and varying combinations of polymers), agent loading, and/orselection of excipients to produce the desired effect.

[0045] As used herein, the term “a” or “an” refers to one or more.

[0046] As used herein, “sufficient bisphosphonate compound to modify therelease profile of the biologically active agent from the biocompatiblepolymer” means that amount of bisphosphonate compound which modifies therelease profile of the biologically active agent from the biocompatiblepolymer which occurs when the sustained release composition does notinclude a bisphosphonate compound.

[0047] “Modifies the release profile” as that term is used herein refersto a prolongation of the period in which a therapeutic amount of thebiologically active agent is released from the biocompatible polymer. Ithas also been observed that the initial release of biologically activeagent can be reduced when a bisphosphonate is present in the sustainedrelease composition.

[0048] A modification of the release profile can be confirmed byappropriate pharmacokinetic monitoring of the patient's serum for thepresence of the biologically active agent or pharmacodynamic monitoringof the patient to monitor the therapeutic effects of the agent upon thepatient. For example, specific antibody testing, as is well known in theart, can be used to determine the concentration of certain biologicallyactive agents in the patient's serum. An example of such testing isdescribed herein for erythropoietin. Further, the therapeutic effect ofthe biologically active agent can be determined by monitoring thepharmocodynamic effects of the biologically active agent. For example,determination of the patient's hematocrit in response to administrationof erythropoeitin, as described herein. Methods of monitoringpharmacodynamic effects can be selected based upon the biologicallyactive agent being administered using widely available techniques.

[0049] As used herein, a “therapeutically effective amount”,“prophylactically effective amount” or “diagnostically effective amount”is the amount of the sustained release composition needed to elicit thedesired biological response following administration.

[0050] The polymers of the invention are biocompatible. Suitablebiocompatible polymers, can be either biodegradable or non-biodegradablepolymers or blends or copolymers thereof, as described herein.

[0051] Suitable biocompatible polymers, can be either biodegradable ornon-biodegradable polymers or blends or copolymers thereof, as describedherein. A polymer is biocompatible if the polymer and any degradationproducts of the polymer are non-toxic to the recipient and also possessno significant deleterious or untoward effects on the recipient's body,such as an immunological reaction at the injection site.“Biodegradable”, as defined herein, means the composition will degradeor erode in vivo to form smaller chemical species. Degradation canresult, for example, by enzymatic, chemical and physical processes.Suitable biocompatible, biodegradable polymers include, for example,poly(lactides), poly(glycolides), poly(lactide-co-glycolides),poly(lactic acid)s, poly(glycolic acid)s, polycarbonates,polyesteramides, polyanydrides, poly(amino acids), polyorthoesters,poly(dioxanone)s, poly(alkylene alkylate)s, copolymers or polyethyleneglycol and polyorthoester, biodegradable polyurethane, blends thereof,and copolymers thereof.

[0052] Suitable biocompatible, non-biodegradable polymers includenon-biodegradable polymers selected from the group consisting ofpolyacrylates, polymers of ethylene-vinyl acetates and other acylsubstituted cellulose acetates, non-degradable polyurethanes,polystyrenes, polyvinylchloride, polyvinyl flouride, poly(vinylimidazole), chlorosulphonate polyolefins, polyethylene oxide, blendsthereof, and copolymers thereof.

[0053] Acceptable molecular weights for polymers used in this inventioncan be determined by a person of ordinary skill in the art taking intoconsideration factors such as the desired polymer degradation rate,physical properties such as mechanical strength, and rate of dissolutionof polymer in solvent. Typically, an acceptable range of molecularweight is of about 2,000 Daltons to about 2,000,000 Daltons.

[0054] In a particular embodiment, the polymer is biodegradable polymeror copolymer. In a more preferred embodiment, the polymer is apoly(lactide-co-glycolide)(hereinafter “PLG”). The PLG can have alactide:glycolide ratio, for example, of about 10:90, 25:75, 50:50,75:25 or 90:10 and a molecular weight of about 5,000 Daltons to about70,000 Daltons.

[0055] The term “biologically active agent,” as used herein, is anagent, or its pharmaceutically acceptable salt, which when released invivo, possesses the desired biological activity, for exampletherapeutic, diagnostic and/or prophylactic properties in vivo. It isunderstood that the term includes stabilized biologically active agentsas described herein.

[0056] Examples of suitable biologically active agents include proteinssuch as immunoglobulins, antibodies, cytokines (e.g., lymphokines,monokines, chemokines), interleukins, interferons, erythropoietin,nucleases, tumor necrosis factor, colony stimulating factors, insulin,enzymes (e.g. superoxide dismutase, plasminogen activator, etc.), tumorsuppressors, blood proteins, hormones and hormone analogs (e.g., growthhormone, adrenocorticotropic hormone, and luteinizing hormone releasinghormone (LHRH)), vaccines (e.g., tumoral, bacterial and viral antigens),antigens, blood coagulation factors; growth factors; peptides such asprotein inhibitors, protein antagonists, and protein agonists; nucleicacids, such as antisense molecules; oligonucleotides; and ribozymes.Small molecular weight agents suitable for use in the invention include,antitumor agents such as bleomycin hydrochloride, carboplatin,methotrexate and adriamycin; antibiotics such as gentamicin,tetracycline hydrochloride and ampicillin; antipyretic, analgesic andanti-inflammatory agents; antitussives and expectorants such asephedrine hydrochloride, methylephedrine hydrochloride, noscapinehydrochloride and codeine phosphate; sedatives such as chlorpromazinehydrochloride, prochlorperazine hydrochloride and atropine sulfate;muscle relaxants such as tubocurarine chloride; antiepileptics such assodium phenytoin and ethosuximide; antiulcer agents such asmetoclopramide; antidepressants such as clomipramine; antiallergicagents such as diphenhydramine; cardiotonics such as theophillol;antiarrhythmic agents such as propranolol hydrochloride; vasodilatorssuch as diltiazem hydrochloride and bamethan sulfate; hypotensivediuretics such as pentolinium and ecarazine hydrochloride; antidiureticagents such as metformin; anticoagulants such as sodium citrate andsodium heparin; hemostatic agents such as thrombin, menadione sodiumbisulfite and acetomenaphthone; antituberculous agents such asisoniazide and ethanbutol; hormones such as predniso lone sodiumphosphate and methimazole; antipsychotic agents such as risperidone; andnarcotic antagonists such as nalorphine hydrochloride.

[0057] In one embodiment, the biologically active agent is stabilized.The biologically active agent can be stabilized against degradation,loss of potency and/or loss of biological activity, all of which canoccur during formation of the sustained release composition having thebiologically active agent dispersed therein, and/or prior to and duringin vivo release of the biologically active agent. In one embodiment,stabilization can result in a decrease in the solubility of thebiologically active agent, the consequence of which is a reduction inthe initial release of biologically active agent, in particular, whenrelease is from a sustained release composition. In addition, the periodof release of the biologically active agent can be prolonged.

[0058] Stabilization of the biologically active agent can beaccomplished, for example, by the use of a stabilizing agent or aspecific combination of stabilizing agents. The stabilizing agent can bepresent in the mixture. “Stabilizing agent” as that term is used herein,is any agent which binds or interacts in a covalent or non-covalentmanner or is included with the biologically active agent. Stabilizingagents suitable for use in the invention are described in U.S. Pat. Nos.5,716,644, 5,674,534, 5,654,010, 5,667,808, and 5,711,968, andco-pending U.S. patent application Ser. No. 08/934,830 to Burke et al.,filed on Sep. 22, 1997 and Ser. No. 09/104,549 to Burke, filed on Jun.25, 1998 the entire teachings of which are incorporated herein byreference.

[0059] For example, a metal cation can be complexed with thebiologically active agent, or the biologically active agent can becomplexed with a polycationic complexing agent such as protamine,albumin, spermidine and spermine, or associated with a “salting-out”salt. In addition, a specific combination of stabilizing agents and/orexcipients may be needed to optimize stabilization of the biologicallyactive agent.

[0060] Suitable metal cations include any metal cation capable ofcomplexing with the biologically active agent. A metal cation-stabilizedbiologically active agent, as defined herein, comprises a biologicallyactive agent and at least one type of metal cation wherein the cation isnot significantly oxidizing to the biologically active agent. In aparticular embodiment, the metal cation is multivalent, for example,having a valency of +2 or more. It is preferred that the metal cation becomplexed to the biologically active agent.

[0061] Suitable stabilizing metal cations include biocompatible metalcations. A metal cation is biocompatible if the cation is non-toxic tothe recipient, in the quantities used, and also presents no significantdeleterious or untoward effects on the recipient's body, such as asignificant immunological reaction at the injection site. Thesuitability of metal cations for stabilizing biologically active agentsand the ratio of metal cation to biologically active agent needed can bedetermined by one of ordinary skill in the art by performing a varietyof stability indicating techniques such as polyacrylamide gelelectrophoresis, isoelectric focusing, reverse phase chromatography, andHPLC analysis on particles of metal cation-stabilized biologicallyactive agents prior to and following particle size reduction and/orencapsulation. The molar ratio of metal cation to biologically activeagent is typically between about 1:2 and about 100:1, preferably betweenabout 2:1 and about 12:1.

[0062] Examples of stabilizing metal cations include, but are notlimited to, K⁺, Zn⁺², Mg⁺² and Ca⁺². Stabilizing metal cations alsoinclude cations of transition metals, such as Cu⁺². Combinations ofmetal cations can also be employed.

[0063] The biologically active agent can also be stabilized with atleast one polycationic complexing agent. Suitable polycationiccomplexing agents include, but are not limited to, protamine, spermine,spermidine and albumin. The suitability of polycationic complexingagents for stabilizing biologically active agents can be determined byone of ordinary skill in the art in the manner described above forstabilization with a metal cation. An equal weight ratio of polycationiccomplexing agent to biologically active agent is suitable.

[0064] Further, excipients can be added to maintain the potency of thebiologically active agent over the duration of release and modifypolymer degradation. The excipients can be added to the dispersed systemwhich is then atomized or can be added to the mixture which is subjectedto fragmenting either before or after fragmentation of the driedsubstance to achieve particles of biologically active agent. Suitableexcipients include, for example, carbohydrates, amino acids, fattyacids, surfactants, and bulking agents, and are known to those skilledin the art. An acidic or a basic excipient is also suitable. The amountof excipient used is based on ratio to the biologically active agent, ona weight basis. For amino acids, fatty acids and carbohydrates, such assucrose, trehalose, lactose, mannitol, dextran and heparin, the ratio ofcarbohydrate to biologically active agent, is typically between about1:10 and about 20:1. For surfactants the ratio of surfactant tobiologically active agent is typically between about 1:1000 and about2:1. Bulking agents typically comprise inert materials. Suitable bulkingagents are known to those skilled in the art.

[0065] The excipient can also be a metal cation component which isseparately dispersed within the polymer matrix. This metal cationcomponent acts to modulate the release of the biologically active agentand is not complexed with the biologically active agent. The metalcation component can optionally contain the same species of metalcation, as is contained in the metal cation stabilized biologicallyactive agent, if present, and/or can contain one or more differentspecies of metal cation. The metal cation component acts to modulate therelease of the biologically active agent from the polymer matrix of thesustained release composition and can enhance the stability of thebiologically active agent in the composition. A metal cation componentused in modulating release typically comprises at least one type ofmultivalent metal cation. Examples of metal cation components suitableto modulate release include or contain, for example, Mg(OH)₂, MgCO₃(such as 4MgCO₃.Mg(OH)₂0.5H₂O), MgSO₄, Zn(OAc)₂, Mg(OAc)₂, ZnCO₃ (suchas 3Zn(OH)₂2ZnCO₃)ZnSO₄, ZnCl₂, MgCl₂, CaCO₃, Zn₃(C₆H₅O₇)₂ andMg₃(C₆H₅O₇)₂. A suitable ratio of metal cation component to polymer isbetween about 1:99 to about 1:2 by weight. The optimum ratio dependsupon the polymer and the metal cation component utilized. A polymermatrix containing a dispersed metal cation component to modulate therelease of a biologically active agent from the polymer matrix isfurther described in U.S. Pat. No. 5,656,297 to Bernstein et al. andco-pending U.S. patent application Ser. No. 09/056,566 filed on Apr. 7,1998, the teachings of both of which are incorporated herein byreference in their entirety.

[0066] The invention described herein also relates to pharmaceuticalcompositions suitable for use in the invention. In one embodiment, thepharmaceutical composition comprises a sustained release compositioncomprising a biocompatible polymer having an effective amount of abiologically active agent incorporated therein, and an amount ofbisphosphonate compound sufficient to modify the release profile of thebiologically active agent from the sustained release composition.

[0067] In one embodiment, the bisphosphonate compound can beco-incorporated into the sustained release composition comprising thebiocompatible polymer and the biologically active agent incorporatedtherein.

[0068] In another embodiment, the pharmaceutical composition comprisesthe sustained release composition comprising a first biocompatiblepolymer having incorporated therein an effective amount of abiologically active agent and a second biocompatible polymer havingincorporated therein an amount of bisphosphonate which modifies therelease profile of the biologically active agent from the first polymer.In a particular embodiment, the first and second polymers are the sametype of polymer. In another embodiment, the first and second polymersare different.

[0069] In yet another embodiment, the bisphosphonate compound can bepresent in the pharmaceutical composition in an unencapsulated state.For example, the bisphosphonate compound can be comingled with thesustained release composition. In one embodiment, the bisphosphonate canbe solubilized in the vehicle used to deliver the pharmaceuticalcomposition. Alternatively, the bisphosphonate compound can be presentas a solid suspended in an appropriate vehicle useful for delivering thepharmaceutical composition. Further, the bisphosphonate can be presentas a powder which is comingled with the sustained release composition.

[0070] The present invention also relates to a composition for thesustained release of bisphosphonates. The sustained release compositioncomprises a biocompatible polymer matrix having a therapeuticallyeffective amount of bisphosphonate incorporated therein. Further, theinvention relates to a method for the sustained release in vivo of abisphosphonate compound comprising administering to a subject in need oftreatment a therapeutically effective amount of a sustained releasecomposition comprising a biocompatible polymer and a bisphosponatecompound.

[0071] In a particular embodiment, administration of the sustainedrelease composition comprising a biocompatible polymer and abisphosphonate can be to a joint, for example, the articular space of ajoint. For example, the sustained release composition can beadministered to the articular space of the knee, shoulder, ankle, hipetc . . .

[0072] The sustained release composition of the invention comprising abiocompatible polymer and a bisphosphonate compound can be used for thetreatment of diseases associated with bone resorption or jointinflammation. For example, the sustained release composition having abiocompatible polymer and a bisphosphonate compound incorporated thereincan be suitable for use as a treatment for rheumatoid arthritis,osteoporosis or Paget's disease.

[0073] A number of methods are known by which sustained releasecompositions (polymer/active agent matrices) can be formed. In many ofthese processes, the material to be encapsulated is dispersed in asolvent containing a wall forming material. At a single stage of theprocess, solvent is removed from the microparticles and thereafter themicroparticle product is obtained.

[0074] Methods for forming a composition for the sustained release ofbiologically active agent are described in U.S. Pat. No. 5,019,400,issued to Gombotz et al., and issued U.S. Pat. No. 5,922,253 issued toHerbert et al. the teachings of which are incorporated herein byreference in their entirety.

[0075] In this method, a mixture comprising a biologically active agent,a biocompatible polymer and a polymer solvent is processed to createdroplets, wherein at least a significant portion of the dropletscontains polymer, polymer solvent and the active. These droplets arethen frozen by a suitable means. Examples of means for processing themixture to form droplets include directing the dispersion through anultrasonic nozzle, pressure nozzle, Rayleigh jet, or by other knownmeans for creating droplets from a solution.

[0076] Means suitable for freezing droplets include directing thedroplets into or near a liquified gas, such as liquid argon or liquidnitrogen to form frozen microdroplets which are then separated from theliquid gas. The frozen microdroplets are then exposed to a liquid orsolid non-solvent, such as ethanol, hexane, ethanol mixed with hexane,heptane, ethanol mixed with heptane, pentane or oil.

[0077] The solvent in the frozen microdroplets is extracted as a solidand/or liquid into the non-solvent to form a polymer/active agent matrixcomprising a biocompatible polymer and a biologically active agent.Mixing ethanol with other non-solvents, such as hexane, heptane orpentane, can increase the rate of solvent extraction, above thatachieved by ethanol alone, from certain polymers, such aspoly(lactide-co-glycolide) polymers.

[0078] A wide range of sizes of sustained release compositions can bemade by varying the droplet size, for example, by changing theultrasonic nozzle diameter. If the sustained release composition is inthe form of microparticles, and very large microparticles are desired,the microparticles can be extruded, for example, through a syringedirectly into the cold liquid. Increasing the viscosity of the polymersolution can also increase microparticle size. The size of themicroparticles which can be produced by this process ranges, forexample, from greater than about 1000 to about 1 micrometers indiameter.

[0079] Yet another method of forming a sustained release composition,from a suspension comprising a biocompatible polymer and a biologicallyactive agent, includes film casting, such as in a mold, to form a filmor a shape. For instance, after putting the suspension into a mold, thepolymer solvent is then removed by means known in the art, or thetemperature of the polymer suspension is reduced, until a film or shape,with a consistent dry weight, is obtained.

[0080] A further example of a conventional microencapsulation processand microparticles produced thereby is disclosed in U.S. Pat. No.3,737,337, incorporated by reference herein in its entirety, wherein asolution of a wall or shell forming polymeric material in a solvent isprepared. The solvent is only partially miscible in water. A solid orcore material is dissolved or dispersed in the polymer-containingmixture and, thereafter, the core material-containing mixture isdispersed in an aqueous liquid that is immiscible in the organic solventin order to remove solvent from the microparticles.

[0081] Another example of a process in which solvent is removed frommicroparticles containing a substance is disclosed in U.S. Pat. No.3,523,906, incorporated herein by reference in its entirety. In thisprocess a material to be encapsulated is emulsified in a solution of apolymeric material in a solvent that is immiscible in water and then theemulsion is emulsified in an aqueous solution containing a hydrophiliccolloid. Solvent removal from the microparticles is then accomplished byevaporation and the product is obtained.

[0082] In still another process as shown in U.S. Pat. No. 3,691,090,incorporated herein by reference in its entirety, organic solvent isevaporated from a dispersion of microparticles in an aqueous medium,preferably under reduced pressure.

[0083] Similarly, the disclosure of U.S. Pat. No. 3,891,570,incorporated herein by reference in its entirety, shows a method inwhich solvent from a dispersion of microparticles in a polyhydricalcohol medium is evaporated from the microparticles by the applicationof heat or by subjecting the microparticles to reduced pressure.

[0084] Another example of a solvent removal process is shown in U.S.Pat. No. 3,960,757, incorporated herein by reference in its entirety.

[0085] Tice et al., in U.S. Pat. No. 4,389,330, describe the preparationof microparticles containing an active agent by a method comprising: (a)dissolving or dispersing an active agent in a solvent and dissolving awall forming material in that solvent; (b) dispersing the solventcontaining the active agent and wall forming material in acontinuous-phase processing medium; (c) evaporating a portion of thesolvent from the dispersion of step (b), thereby forming microparticlescontaining the active agent in the suspension; and (d) extracting theremainder of the solvent from the microparticles.

[0086] Without being bound by a particular theory it is believed thatthe release of the biologically active agent can occur by two differentmechanisms. First, the biologically active agent can be released bydiffusion through aqueous filled channels generated in the polymermatrix, such as by the dissolution of the biologically active agent, orby voids created by the removal of the polymer solvent during thepreparation of the sustained release composition. A second mechanism isthe release of the biologically active agent, due to degradation of thepolymer. The rate of degradation can be controlled by changing polymerproperties that influence the rate of hydration of the polymer. Theseproperties include, for instance, the ratio of different monomers, suchas lactide and glycolide, comprising a polymer; the use of the L-isomerof a monomer instead of a racemic mixture; and the molecular weight ofthe polymer. These properties can affect hydrophilicity andcrystallinity, which control the rate of hydration of the polymer.

[0087] By altering the properties of the polymer, the contributions ofdiffusion and/or polymer degradation to biologically active agentrelease can be controlled. For example, increasing the glycolide contentof a poly(lactide-co-glycolide) polymer and decreasing the molecularweight of the polymer can enhance the hydrolysis of the polymer andthus, provides an increased biologically active agent release frompolymer erosion.

[0088] The composition of this invention can be administered in vivo,for example, to a human, or to an animal, orally, or parenterally suchas by injection, implantation (e.g., subcutaneously, intramuscularly,intraperitoneally, intracranially, and intradermally), administration tomucosal membranes (e.g., intranasally, intravaginally, intrapulmonary,buccally or by means of a suppository), or in situ delivery (e.g., byenema or aerosol spray) to provide the desired dosage of antigen orlabile agent based on the known parameters for treatment with theparticular agent of the various medical conditions.

[0089] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the scope of the inventionencompassed by the appended claims.

[0090] Exemplifications

[0091] Materials and Methods

[0092] In Ivo Testing

[0093] Male Sprague-Dawley Rats, weighing between 350 to 450 grams(Charles River Laboratories, Inc.) were used in the studies describedbelow following acclimation in standard animal housing for at leastseven days. Animals were treated with cyclosporin (Sandimmune, Sandoz;CS) 5 mg/kg ip daily for days 0-14 post administration of the sustainedrelease composition and bisphosphonate, and then 3 times per weekthereafter. In some instances, cyclosporin treatment was omitted for atleast two non-consecutive days during the initial 0-14 day treatment.Administration of the EPO-containing microparticles and bisphosphonatecompound is described in detail below.

[0094] Preparation of Epo-Containing Microparticles

[0095] Microparticles containing recombinant human Erythropoietin (EPO)were made following the procedure described in U.S. Pat. No. 5,716,644issued on Feb. 10, 1998 to Zale et al., the entire content of which ishereby incorporated by reference. Specifically, the EPO-containingmicroparticles were prepared using a polymer purchased from Alkermes,Inc. of Cincinnati, Ohio having Cat No.5050DL2A which is apoly(lactide-co-glycolide) 10 kD polymer having a lactide/glycolideratio of 50:50. The biosphosphonate-containing microparticles wereprepared using a poly(lactide-co-glycolide) 25 kD polymer having alactide/glycolide ratio of 50:50 also available from Alkermes, Inc. ofCincinnati, Ohio and having Cat. No. 5050DL3A. The EPO was obtained fromJohnson & Johnson, New Brunswick, N.J. and stabilized prior toencapsulation as described in U.S. Pat. No. 5,716,644 using an EPOloading of about 1.6% w/w of the total weight of stabilized EPO in themicroparticles.

[0096] Preparation of Bisphosphonate-Containing Microparticles

[0097] Bisphosphonate-containing microparticles having both a 1% and2.5% w/w of the final weight of the microparticle theoretical load ofthe indicated bisphosphonate were prepared using apoly(lactide-co-glycolide) Cat. No. 5050DL3 earlier described having alactide:glycolide ratio of 50:50.

[0098] Briefly, the bisphosphonates are soluble in water and insolublein organics, making phase separation a suitable method for use inpreparing the bisphosphonate-containing microparticles. First 212 mg ofthe poly(D,L-lactide-co-glycolide), with a molecular weight of about 25kD available from Alkermes, Inc. of Cincinnati, Ohio, as Cat. No.5050DL3A was weighed out and dissolved in methylene chloride at aconcentration of about 5.9% W/V. 90 mg pamidronate disodium in 375 mgmannitol (available as AREDIA 90®) was weighed out and dissolved inabout 3 g water. The encapsulate solution was then added to the polymersolution, and probe sonicated using pulses for about 1 minute togenerate an extremely fine water-in-oil (W/O) emulsion.

[0099] The resulting emulsion was charged to a 350 mL glass reactor. Thestir speed was set to about 1000 RPM. The coacervation agent, DowCorning 360 Fluid, 350 cs, was slowly added by peristaltic pump to thestirring W/O emulsion to induce phase separation. Dow Coming 360 Fluidaddition was halted when a 1:1 ratio of fluid to methylene chlorideratio had been achieved. Then the bottom stopcock of the reactor wasopened in order to gravity feed embryonic microparticles into a heptanequench. After stirring for about 2 hours in the heptane quench, thehardened microparticles were isolated by filtration and allowed to dryin an ambient temperature vacuum chamber overnight. The product was thencollected and weighed.

EXAMPLE 1

[0100] Pharmacological Effects of Bisphosphonate-ContainingMicroparticles on Epo Release from Epo-Containing Micro FollowingCo-Administration

[0101] The Phamacokinetic (PK)/Pharmacodynamic (PD) response to EPOreleased from EPO-containing microparticles when co-administered withbisphosphonate-containing microparticles in vivo to male Sprague-Dawleyrats was determined.

[0102] Microparticle Administration

[0103] Animals were anesthetized with 5% halothane. Each animal wasshaved and the back swabbed with alcohol. Microparticles wereresuspended using 0.75 mL vehicle (3% carboxymethylcellulose, 0.1% Tween20, 0.9% NaCl, pH ˜6). The microparticles were injected into aninterscapular site using a 21 guage thinwall needle attached to a 1 mLsyringe. Animals were dosed to receive a total of 10,000 U EPO incombination with a total of 2.5 mg of the indicated bisphosphonate. Theamount of bisphosphonate-containing microparticles needed was determinedbased on the theoretical load of bisphosphonate in the microparticleformulation. For example, 100 mg of bisphosphonate-containingmicroparticles have a 2.5% theoretical load of bisphosphonate resultedin administration of 25 mg. Animals were followed for 47 days postimplantation, except for Group G (Alendronate), which was followed for57 days. The microparticles mixed and administered as one injection.

[0104] Bisphosphonate-containing microparticles having a 2.5%theoretical load of bisphosphonate were prepared as described above witha 2.5% theoretical load using the following bisphosphonate comounds:pamidronate (AREDIA®, (3-amino-1-hydroxypropylidene)bispnosphonic acid)disodium salt), etidronate ((1-hydroxyethyledene)bisphophonic acid)disodium salt, DIDRONEL®), tiludronate([[(4-chlorophenyl)thio]methylene]bisphosphonic acid disodium salt,SKELID®), risedronate([1-hydroxy-2-(3-pyridinyl)ethylidene]bisphosphonic acid) monosodiumsalt, ACTONEL®), and alendronate((4-amino-1-hydroxybutylidene)bisphosphonic acid) monosodium salt,FOSAMAX®). In addition, microparticles having no bisphosphonate compoundincorporated therein (placebo) were prepared following the methodoutlined.

[0105] Sample Collection Timepoints (days)

[0106] pre-bleed, 1,2,5,8,12,15,19,22, 26,29,33,36,40,42,47, 50, 54, &57 TABLE 1 Bisphosphonate # of Animal EPO Dose Bisphophonate Dose Groupper Group (Units) Compound (mg) A 4 10,000 — — B 4 10,000 Placebo —Microparticles C 4 10,000 Pamidronate 2.5 mg (AREDIA ®) D 4 10,000Etidronate 2.5 mg (DIDRONEL ®) E 4 10,000 Tiludronate 2.5 mg (SKELID ®)F 4 10,000 Risedronate 2.5 mg (ACTONEL ®) G 4 10,000 Alendronate 2.5 mg(FOSAMAX ®)

[0107] Serum Evaluation

[0108] Serum samples (40 μL) were collected via tail vein on thefollowing days relative to microparticle administration: pre-bleed, 1,2, 5, 8, 12, 15, 19, 22, 26, 29, 33, 36, 40, 42, 47, 50, 54 and 57.After clotting, the samples were centrifuged and frozen at −70° C. SerumEPO levels were quantitated by ELISA (R&D Systems, Minneapolis, Minn.Cat. No. DEPOO). The results are presented graphically in FIG. 1.

[0109] Hematocrits were evaluated manually following centrifugation for5 minutes at 8000 rpm (on four animals per group) using a capillarytube. Hematocrits were also determined at the following intervalsrelative to microparticle administration: pre-bleed, 1, 2, 5, 8, 12, 15,19, 22, 26, 29, 33, 36, 40, 42, 47, 50, 54 and 57. The results arepresented graphically in FIG. 2.

[0110] Animals were observed on a regular basis for any signs ofabnormal behavior. Injection sites were observed on a regular basis forinduration, weeping or erythema. Sites were observed on a weekly basisfor blanching. Body weights were taken and recorded at each samplecollection timepoint.

[0111] The experiment was terminated once EPO serum levels had fallenbelow the limit of quantitation.

[0112] Results

[0113] The dose normalized data from ELISA testing of serum EPO levelsare shown in FIG. 1. The data show that each of the bisphosphonatecompounds tested enhanced the release of EPO from EPO-containingmicroparticles in comparison to the group receiving only EPO-containingmicroparticles. However, alendronate alone was shown to significantlyaffect the duration of EPO release from EPO-containing microparticles incomparison to groups receiving placebo microparticles co-administeredwith EPO-containing microparticles. That is, the animals of Group G hadmeasurable levels of serum EPO at day 29 (23.17±8.22). This wassignificant in comparison to the placebo treated controls which werebelow the limit of quantitation at p<0.01. Further, a significantreduction in burst was seen in the Group C animals (Cmax: 2072.73±437.8)when compared to Group B animals (Cmax: 3989.56±883.47, p<0.05).Although the pamidronate treatment (Group C), increased the duration ofrelease of detectable levels of EPO, it was not significantly differentfrom the placebo-treated group (group B).

[0114] Hematocrits in 4 out of 5 of the treated groups rose above thatof the control groups A and B. Further, it was observed for Group G(alendronate) that the hematocrits remained significantly higher thancontrols past day 36 (FIG. 2). Day 36 is the last day serum levels ofEPO were positive. The Alendronate treated group demonstrated higherhematocrits out to Day 47 which is the last day measured for controlGroups A and B, demonstrating that the pharmacodynamic (PD) responseextended out beyond pharmacokinetic (PK) response. In addition, attimepoints 26, 33 and 36 days, the hematocrits in the the Group Canimals (pamidronate treated) were also significantly higher thancontrols (p<0.05). Following day 26, the hematocrits of all groups, withthe exception of Group G, had reached baseline or near baseline levels.That is, Group G had significantly higher hematocrits than controls atall time points between day 26 and 47 (p<0.05) and did not approachbaseline until day 57. Finally, comparing 60% hematocrits, thealendronate extended the PD response to EPO from day 32 in the placebocontrol group (B) to day 49. This is a 17-day enhancement usingalendronate.

[0115] Pamidronate disodium (AREDIA®) in mannitol was dosed at 1 mg andadmixed with EPO-containing microparticles.

[0116] In the group that received pamidronate in the vehicle (Group E)there was also an increase in duration of release. Group E also showed asignificant elevation of release in the day 12 to day 15 timepointsrelative to other groups. This enhanced release can be useful inclinical settings where tailored release is desired.

[0117] The use of pamidronate in vehicle (group E) also enhanced EPOPK/PD

EXAMPLE 2

[0118] Co-Administration of Epo-Containing Microparticles withAlendronate-Containing Microparticles at Varying Doses

[0119] This example compares the PK/PD response to EPO released fromEPO-containing microparticles when co-administered with various doses ofalendronate-containing microparticles.

[0120] Materials and Methods

[0121] EPO-containing microparticles, alendronate-containingmicroparticles and placebo microparticles were prepared as described inExample 1. Alendronate-containing microparticles were prepared at aloading of 1.0% and 2.5% (theoretical). Microparticle administration,sample collection and sample analysis were as described in Example 1 andare summarized in Table 2. Sample collection timepoints were pre-bleed,1, 2, 5, 8, 12, 15, 19, 22, 26, 29, 33, 36, 40. TABLE 2 Number ofAlendronate Animals Dose (mg) Placebo per (theoretical load × mg DoseGroup Group Epo Dose (U) μparticles = dose) (mg) I 4 10,000 U — 100 mg II 4 10,000 U — 50 mg III 4 10,000 U — 25 mg IV 4 — 2.5 mg — (2.5% × 100mg μparticles) V 4 10,000 U 2.5 mg — (2.5% × 100 mg μparticles) VI 410,000 U 1.25 mg — (2.5% × 50 mg μparticles) VII 4 10,000 U 0.625 mg —(2.5% × 25 mg μparticles) VIII 4 10,000 U 1.0 mg — (1.0% × 100 mgμparticles) IX 4 10,000 U 0.5 mg — (1.0% × 50 mg μparticles) X 4 10,000U 0.25 mg — (1.0% × 25 mg μparticles)

[0122] Results:

[0123] Serum ELISA data show a clear dose response in animals thatreceived the 1% and 2.5% alendronate encapsulated microspheres. FIG. 3shows the phannacokinetic profile over the study period out to 36 days.Serum EPO levels in animals of Groups I, II and III, receiving onlyEPO-containing microparticles and placebo microparticles (nobisphosphonate) at 100, 50 and 25 mg doses were not measurable after day22. Groups VIII, IX and X which received 1% alendronate-containingmicroparticles with a total dose of 1.0, 0.5 and 0.25 mg declined by day29. However, serum EPO levels in Groups V, VI and VII which received2.5% loaded alendronate-containing microparticles for a total of 2.5,1.25 and 0.625 mg of alendronate were detectable out to at least day 34.

[0124] Table 3 summarizes Cmax data for this study. The Cmax for GroupsV and VI receiving 100 or 50 mg of the 2.5% loadedalendronate-containing microparticles for a total of 2.5 and 1.25 mg,respectively were significantly lower than the dose matched placeboGroups I and II (p<0.05). Cmax was not significantly suppressed forGroup VII compared to the corresponding placebo control. Steady statelevels (Day 5 to Day 29) for Groups VI and VII were increased from 48 to132 and 56 to 146 relative to their controls (p<0.05). Groups VIII, IXand X (1% loaded microparticles) showed steady state serum EPO increasesof 48 to 84 and 56 to 111 at the 25 and 50 mg doses, however thesedifferences were not statistically significant compared to the placebogroups at the same doses of blank microparticles. TABLE 3 Group CmaxTmax Ave Steady Stat 100 mg I EPO + 100 mg Placebo 3764.79 ± 425.06 181.17 ± 17.19 V EPO + 100 mg 2.5% Alendronate 2003.31 ± 308.80 1 116.17± 24.18  VIII EPO + 100 mg 1.0% Alendronate 2966.20 ± 530.14 1 103.60 ±7.47  50 mg II EPO + 50 mg Placebo 2968.51 ± 260.47 1 48.27 ± 14.83 VIEPO + 50 mg 2.5% Alendronate 2538.13 ± 231.93 1 132.41 ± 24.90  IX EPO +50 mg 1.0% Alendronate 2464.98 ± 647.79 1 84.96 ± 35.89 25 mg III EPO +25 mg Placebo 3597.26 ± 247.76 1 56.48 ± 10.09 VII EPO + 25 mg 2.5%Alendronate 2972.05 ± 823.56 1 146.92 ± 36.33  X EPO + 25 mg 1.0%Alendronate  3452.95 ± 1132.84 1 111.79 ± 54.93 

[0125]FIG. 4A shows an enhanced hematocrit response in rats to all threegroups receiving varying doses of the 2.5% alendronatecontaining-microparticles co-administered with EPOcontaining-microparticles (Groups V, VI and VII) when compared to groupsreceiving the corresponding placebo dose of microparticles (FIG. 4C).The duration of hematocrits >60% was increased by at least 6 days in allgroups. The complete enhancement effect on the pharmacodynamics couldnot be determined as a result of termination of the study at day 40.

[0126] The hematocrit levels for Groups V, VI and VII (FIG. 4A) wereover 65% at day 40, significantly higher than controls (56%, P<0.01).FIG. 4B shows the hematocrit levels of the Groups VIII, IX and X treatedwith 100 mg, 50 mg or 25 mg of the 1% alendronate-containingmicroparticles, respectively co-administered with EPOcontaining-microparticles. Amongst Groups VIII, IX and X, only the 100mg dose (Group VIII) was significantly higher at day 40 than thehematocrit value of the group receiving 100 mg of placebo microparticlesco-administered with EPO-containing microparticles.

[0127]FIG. 4C represents hematocrit values obtained following injectionwith placebo microparticless co-administered with EPO-containingmicroparticles. It noted from FIGS. 4A-4C that there were no significantdifferences between hematocrits of theGroups receiving varying doses of2.5% loaded alendronate-containing microparticles, receiving varyingdoses of 1.0% loaded alendronate-containing microparticles and varyingdoses of placebo microparticles when co-administered with EPO-containingmicroparticles.

[0128] Groups IV, V and VI (co-administration of 2.5% loadedalendronate-containing microparticles) as well as Group VIII (100 mgdose of the 1% alendronate-containing microparticles) had palpablemasses at the injection sites at day 40. The study was terminated atthis time to allow recovery of injection sites. There were noraised/inflammed sites in the placebo groups, however, which indicatesthat the inflammation was limited to the alendronate groups. Where therewas a lower dose of microparticles, the size of the swelling was alsosmaller, and in the two lowest doses of alendronate (1% load, 50 and 25mg doses) there were no palpable masses through the skin. Inflammationseen with alendronate groups is likely due to an acute phase responsethat can occur with amine containing bisphosphonates.

EXAMPLE 3

[0129] Effects of Pamidronate Co-Encapsulated with Epo Materials andMethods:

[0130] EPO-containing microparticles were prepared as described inExample 1 using a 40 kD Polymer poly(lactide-co-glycolide) polymerhaving a lactide glycolide ratio of 50:50 (Cat. No. 5050DL4A, Alkermes,Inc., Cinncinnati, Ohio). In addition, EPO-containing microparticles(1.9% theoretical load) having pamidronate co-encapsulated at nominalloads of 1% and 10% (theoretical) were also prepared as described inExample 1 for EPO alone.

[0131] Microparticle administration, sample collection and analysis wereas described in Example 1 and are summarized in Table 4. Samplecollection timepoints were pre-bleed, 1, 2, 6, 9, 13, 16, 20, 23, 27,30, 34, 37, 41, 44, 48 days. TABLE 4 # Animals per Amount of PamidronateGroup Group EPO (% Theoretical Load) X  5* 20,000 U — Y 5 20,000 U  1% Z5 20,000 U 10%

[0132] Results:

[0133] Following an initial peak at nearly 10,000 mU/mL or above, serumEPO levels in all groups decreased sharply until day 6, when asteady-state was reached at approximately 100 mU/ml (FIG. 5). However,serum EPO levels in groups which had been treated with EPO-containingmicroparticles co-encapsulated with 1% pamidronate began to decreasemore rapidly such that the levels were at the assay detection limit byday 16. While serum EPO levels were elevated over controls with themicroparticles containing EPO and 10% pamidronate, levels did not remainabove controls after day 23. The serum EPO steady state level of thegroups compared over day 9 through 27 showed a significant increase(p<0.05) in the group receiving 10% pamidronate-containingmicroparticles (Table 5) compared to the control group. Neither 1% or10% containing pamidronate microparticles lowered EPO burstsignificantly, nor did it extend the steady state levels of EPO longerthan control animals. TABLE 5 EPO-Containing Microparticles EPO, 20,000U EPO, 20,000 U 20,000 U Pamidronate 1% Pamidronate 10% Cmax, mU/mL43090.42 26308.30 31472.63 Tmax, day 1.00 1.00 1.00 AUC 0-1 d 21545.2110718.10 11249.28 mU day/mL AUC 0-3 d, 52655.19 26229.50 24860.95 mUday/mL AUC d33, 97692.89 61599.38 63330.36 mU day/mL AUC (0-1)/ 22.8424.87 22.29 (0-last) % AUC (0-3)/ 56.22 59.59 46.79 (0-last) % Averagesteady 54.12 13.82 125.64 state value (days 9-27):

[0134] Hematocrits were evaluated in the Groups X, Y and Z as a measureof the pharmacodynamic effect of EPO (FIG. 6). No significant effectswere observed in the group receiving 1% bisphosphonate relative tocontrols. When animals received 10% bisphosphonate incorporated withinEPO-containing microparticles, hematocrits showed an upward trend overthe time period between 9 to 37 days. The difference in hematocrits isstatistically different from controls on days 13 and 16 (p<0.05). Thisis consistent with the enhanced PK values for the group receiving 10%bisphosphonate observed between days 13 and 20.

[0135] The bisphosphonate, pamidronate was able to modulate both PK andPD responses to EPO when co-encapsulated into ProLease microspheres at a10% nominal load. In this example, no increase in persistence of EPOresponses was observed. However, the pamidronate did cause an elevationof EPO circulating serum levels for approximately one week. Thisincrease is also reflected in enhanced pharmacodynamic responses.

[0136] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A method for the sustained release in vivo of abiologically active agent comprising administering to a subject in needof treatment an effective amount of a sustained release compositioncomprising a biocompatible polymer having a biologically active agentincorporated therein, and a bisphosphonate wherein the bisphosphonate ispresent in an amount sufficient to modify the release profile of thebiologically active agent from the sustained release composition.
 2. Themethod of claim 1, wherein the bisphosphonate compound isco-incorporated into the sustained release composition.
 3. The method ofclaim 1, wherein the bisphosphonate compound is separately incorporatedinto a second biocompatible polymer.
 4. The method of claim 3, whereinthe second biocompatible polymer is the same as the biocompatiblepolymer of the sustained release composition.
 5. The method of claim 4,wherein the second biocompatible polymer is different from thebiocompatible polymer of the sustained release composition.
 6. Themethod of claim 1, wherein the bisphosphonate compound isunencapusulated but comingled with the sustained release composition. 7.The method of claim 1 wherein the the biocompatible, biodegradablepolymer of the microparticle is selected from poly(lactides),poly(glycolides), poly(lactide-co-glycolides), poly(lactic acid)s,poly(glycolic acid)s, polycarbonates, polyesteramides, polyanydrides,poly(amino acids), polyorthoesters, poly(dioxanone)s, poly(alkylenealkylate)s, copolymers of polyethylene glycol and polyorthoester,polyurethanes, blends thereof, and copolymers thereof.
 8. The method ofclaim 7 wherein the biocompatible, biodegradable polymer is apoly(lactide-co-glycolide).
 9. The method of claim 1 wherein thebisphosphonate is alendronate, risendronate, pamidonate, etidronate,tiludronate or a combination thereof.
 10. The method of claim 1, whereinthe sustained release composition is in the form of microparticles. 11.The method of claim 1 wherein the biologically active agent is aprotein, a peptide or a nucleic acid.
 12. The method of claim 1 whereinthe biologically active agent is a protein.
 13. The method of claim 1wherein the protein is erythropoietin.
 14. A pharmaceutical compositioncomprising: a) a sustained release composition comprising abiocompatible polymer having an effective amount of a biologicallyactive agent incorporated therein; and b) a bisphosphonate compound,wherein the bisphosphonate compound is present in an amount sufficientto modify the release profile of the biologically active agent from thesustained release composition.
 15. The pharmaceutical composition ofclaim 14, wherein the bisphosphonate compound is co-incorporated intothe sustained release composition.
 16. The pharmaceutical composition ofclaim 14, wherein the bisphosphonate compound is separately incorporatedinto a second biocompatible polymer.
 17. The pharmaceutical compositionof claim 16, wherein the second biocompatible polymer is the same as thebiocompatible polymer of the sustained release composition.
 18. Thephamaceutical composition of claim 16, wherein the second biocompatiblepolymer is different from the biocompatible polymer of the sustainedrelease composition.
 19. The pharmaceutical compositin of claim 14,wherein the bisphosphonate compound is unencapusulated but comingledwith the sustained release composition.
 20. The pharmaceuticalcomposition of claim 14 wherein the the biocompatible, biodegradablepolymer of the microparticle is selected from poly(lactides),poly(glycolides), poly(lactide-co-glycolides), poly(lactic acid)s,poly(glycolic acid)s, polycarbonates, polyesteramides, polyanydrides,poly(amino acids), polyorthoesters, poly(dioxanone)s, poly(alkylenealkylate)s, copolymers of polyethylene glycol and polyorthoester,polyurethanes, blends thereof, and copolymers thereof.
 21. Thepharmaceutical composition of claim 20 wherein the biocompatible,biodegradable polymer is a poly(lactide-co-glycolide).
 22. Thepharmaceutical composition of claim 14 wherein the bisphosphonate isalendronate, risendronate, pamidonate, etidronate, tiludronate or acombination thereof.
 23. The pharmaceutical composition of claim 14,wherein the sustained release composition is in the form ofmicroparticles.
 24. The pharmaceutical composition of claim 14, whereinthe biologically active agent is a protein, a peptide or a nucleic acid.25. The pharmaceutical composition of claim 14, wherein the biologicallyactive agent is a protein.
 26. The pharmaceutical composition of claim14, wherein the protein is erythropoietin.